Epithelial cells are considered to be a main target of bleomycin-induced lung injury, which leads to fibrosis in vivo. We studied the characteristics of in vitro bleomycin-induced apoptosis in a mouse lung epithelial (MLE) cell line. Bleomycin caused an increase of reactive oxygen species (ROS) resulting in oxidative stress, mitochondrial leakage, and apoptosis. These were associated with elevated caspase-8 and resultant caspase-9 activity and with upregulation of Fas expression. Glutathione and inhibitors of caspase-8 or caspase-9, but not of FasL, inhibited these effects, suggesting their dependence on ROS, caspase-8 and -9, in a Fas/FasL-independent pathway. However, postbleomycin-exposed MLE cells were more sensitive to Fas-mediated apoptosis. These results demonstrate that the initial bleomycin-induced oxidative stress causes a direct apoptotic effect in lung epithelial cells involving a regulatory role of caspase-8 on caspase-9. Fas represents an amplification mechanism, and not a direct trigger of bleomycin-induced epithelial cell apoptosis.

Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARα, PPARγ, and LXRα. We show it activates the ligand-binding domain of both PPARα and PPARγ, while inhibiting LXRα in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRα, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARα co-activator, PGC1α. The flavonoid activates PPAR response element (PPRE) while suppressing LXRα response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRα-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRα inhibitor.

Background & Aims Hepatitis C virus (HCV) infection affects 3% of the world population and is the leading cause of chronic liver disease worldwide. Current standard of care is effective in only 50% of the patients, poorly tolerated, and associated with significant side effects and viral resistance. Recently, our group and others demonstrated that the HCV lifecycle is critically dependent on host lipid metabolism and that its production is metabolically modulated. Methods The JFH1/Huh7.5.1 full lifecycle model of HCV was used to study the antiviral effects of naringenin on viral replication, assembly, and production. Activation of PPARα was elucidated using GAL4-PPARα fusion reporters, PPRE reporters, qRT-PCR, and metabolic studies. Metabolic results were confirmed in primary human hepatocytes. Results We demonstrate that the grapefruit flavonoid naringenin dose-dependently inhibits HCV production without affecting intracellular levels of the viral RNA or protein. We show that naringenin blocks the assembly of intracellular infectious viral particles, upstream of viral egress. This antiviral effect is mediated in part by the activation of PPARα, leading to a decrease in VLDL production without causing hepatic lipid accumulation in Huh7.5.1 cells and primary human hepatocytes. Long-term treatment with naringenin leads to a rapid 1.4 log reduction in HCV, similar to 1000 U of interferon. During the washout period, HCV levels returned to normal, consistent with our proposed mechanism of action. Conclusions The data demonstrates that naringenin is a non-toxic assembly inhibitor of HCV and that other PPARα agonists play a similar role in blocking viral production. The combination of naringenin with STAT-C agents could potentially bring a rapid reduction in HCV levels during the early treatment phase, an outcome associated with sustained virological response.

Epithelial cell injury and apoptosis are recognised as early features in idiopathic pulmonary fibrosis and bleomycin-induced fibrosis in mice. Telomerase is a known apoptosisalleviating factor. The role of telomerase was studied during bleomycin-induced lung epithelial cell (LEC) apoptosis in vitro in a mouse LEC line, and in vivo in LECs isolated from bleomycintreated mice.The current authors evaluated changes in murine telomerase reverse transcriptase (mTERT) mRNA levels and changes in telomerase activity with the TRAPeze Detection Kit, telomeric length with the TeloTTAGGG Telomere Length Kit, and LEC apoptosis with FACScan and 4,6-diamino-2-phenylindole dihydrochloride stain.There was a significant elevation in mTERT mRNA and a transient 41% increase in telomerase activity 24 h after in vitro bleomycin treatment. At 72 h, telomerase activity had fallen to 26% below levels in untreated cells. Reduction of telomerase activity over time, or by direct inhibition, significantly elevated LEC apoptosis. No change in average telomeric length was noted. In vivo, telomerase activity of LECs from bleomycin-treated mice increased at 7 and 14 days.In conclusion, telomerase activity may play a protective role against robust bleomycin-induced lung epithelial cell apoptosis. Moreover, stabilising telomerase activity may decrease epithelial cell apoptosis and the resulting lung fibrosis.

IFN-γ production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-γ expression in bleomycin-induced lung injury. IFN-γ mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-γ protein levels were increased at 6 days, as was the percentage of LC expressing IFN-γ. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-γ production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-γ production by these cells. To define the role of endogenous IFN-γ in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-γ gene (IFN-γ knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-γ knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-γ production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-γ in IFN-γ knockout mice does not increase pulmonary fibrosis. Endogenous IFN-γ may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.